Auristatin Payload Research Articles

Preclinical Evaluation of STI-8811, a Novel Antibody-Drug Conjugate Targeting BCMA for the Treatment of Multiple Myeloma.

Treatment for patients with multiple myeloma has experienced rapid development and improvement in recent years; however, patients continue to experience relapse, and multiple myeloma remains largely incurable. B-cell maturation antigen (BCMA) has been widely recognized as a promising target for treatment of multiple myeloma due to its exclusive expression in B-cell linage cells and its critical role in the growth and survival of malignant plasma cells. Here, we introduce STI-8811, a BCMA-targeting antibody-drug conjugate (ADC) linked to an auristatin-derived duostatin payload via an enzymatically cleavable peptide linker, using our proprietary C-lock technology. STI-8811 exhibits target-specific binding activity and rapid internalization, leading to G2/M cell-cycle arrest, caspase 3/7 activation, and apoptosis in BCMA-expressing tumor cells in vitro. Soluble BCMA (sBCMA) is shed by multiple myeloma cells into the blood and increases with disease progression, competing for ADC binding and reducing its efficacy. We report enhanced cytotoxic activity in the presence of high levels of sBCMA compared with a belantamab mafodotin biosimilar (J6M0-mcMMAF). STI-8811 demonstrated greater in vivo activity than J6M0-mcMMAF in solid and disseminated multiple myeloma models, including tumor models with low BCMA expression and/or in large solid tumors representing soft-tissue plasmacytomas. In cynomolgus monkeys, STI-8811 was well tolerated, with toxicities consistent with other BCMA-targeting ADCs with auristatin payloads in clinical studies. STI-8811 has the potential to outperform current clinical candidates with lower toxicity and higher activity under conditions found in patients with advanced disease. Significance: STI-8811 is a BCMA-targeting ADC carrying a potent auristatin derivative. We report unique binding properties which maintain potent cytotoxic activity under sBCMA-high conditions that hinder the clinical efficacy of current BCMA-targeting ADC candidates. Beyond disseminated models of multiple myeloma, we observed efficacy in solid tumor models of plasmacytomas with low and heterogenous BCMA expressions at a magnitude and duration of response exceeding that of clinical comparators.

“Negative” Impact: The Role of Payload Charge in the Physicochemical Stability of Auristatin Antibody–Drug Conjugates

Antibody-drug conjugates (ADCs) tend to be less stable than their parent antibodies, which is often attributed to the hydrophobic nature of their drug payloads. This study investigated how the payload charge affects ADC stability by comparing two interchain cysteine ADCs that had matched drug-to-antibody ratios and identical linkers but differently charged auristatin payloads, vcMMAE (neutral) and vcMMAF (negative). Both ADCs exhibited higher aggregation than their parent antibody under shaking stress and thermal stress conditions. However, conjugation with vcMMAF increased the aggregation rates to a greater extent than conjugation with uncharged but more hydrophobic vcMMAE. Consistent with the payload logD values, ADC-vcMMAE showed the greatest increase in hydrophobicity but minor changes in charge compared with the parent antibody, as indicated by hydrophobic interaction chromatography and capillary electrophoresis data. In contrast, ADC-vcMMAF showed a decrease in net charge and isoelectric point along with an increase in charge heterogeneity. This charge alteration likely contributed to a reduced electrostatic repulsion and increased surface activity in ADC-vcMMAF, thus affecting its aggregation propensity. These findings suggest that not only the hydrophobicity of the payload, but also its charge should be considered as a critical factor affecting the stability of ADCs.

Abstract 4476: Novel auristatins with improved tolerability and unique bystander activity profile

Abstract Auristatins are a class of clinically validated payloads used extensively in antibody-drug conjugate (ADC) technology. There are currently 4 FDA approved ADCs utilizing the vedotin drug linker to treat an array of solid tumors and lymphomas and several more in clinical trials. Auristatin payloads have several desirable properties, including strong anticancer cytotoxicity, bystander activity, and induction of immunologic cell death. While auristatin-based ADCs have been remarkably successful, there remain opportunities to further understand and improve payload properties. Bystander activity is one such property that can greatly impact the balance between efficacy and tolerability. Herein, we present a medicinal chemistry investigation into a series of auristatins with similar tubulin binding and biochemical potency that differ in lipophilicity and ability to cross the cell membrane. This design enabled us to focus on and characterize the impact of bystander activity while maintaining all other parameters. Ultimately, this effort resulted in ADCs with similar on-target in vitro cytotoxicity but differed in the extent of off-target bystander activity. Evaluation of in vivo efficacy, in vivo bystander activity, and rodent tolerability afforded a lead drug linker that was well-tolerated as an ADC and exhibited strong antitumor growth delay. Citation Format: Philip N. Moquist, Nicole M. Eng-Duncan, Tim Bovee, Katie Snead, Jenn Wright, Kaleb Smith, Teri Blevins, Brittney Blackburn, Michelle Ulrich, Forgivemore Magunda, Jessica Simmons, Peter D. Senter, Svetlana O. Doronina. Novel auristatins with improved tolerability and unique bystander activity profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4476.

Trastuzumab-MMAU Antibody-Auristatin Conjugates: Valine-Glucoserine Linker with Stabilized Maleimide Conjugation Improves In Vivo Efficacy and Tolerability.

Antibody-drug conjugates (ADCs) have shown impressive clinical activity with approval of many agents in hematological and solid tumors. However, challenges remain with both efficacy and safety of ADCs. This study describes novel trastuzumab-auristatin conjugates with the hydrophilic MMAE prodrug MMAU, and optimization of a glycopeptide linker leading to a wider therapeutic window. Trastuzumab was conjugated with auristatin payloads via a series of linkers using a stabilized maleimide handle. The ADCs were characterized in vitro and their relative in vivo anti-tumor efficacies were assessed in HER2+ xenograft models. Relative linker stabilities and the mechanism of linker cleavage were studied using in vitro assays. Toxicity and toxicokinetics of the best performing ADC were evaluated in cynomolgus monkey (cyno). The trastuzumab-MMAU ADC with stabilized glycopeptide linker showed maleimide stabilization and higher resistance to cleavage by serum and lysosomal enzymes compared to a valine-citrulline conjugated trastuzumab ADC (trastuzumab-vc-MMAE). A single dose of 1 or 2 mg/kg of trastuzumab-MMAU at drug-to-antibody ratios (DAR) of 8 and 4 respectively resulted in xenograft tumor growth inhibition, with superior efficacy to trastuzumab-vc-MMAE. Trastuzumab-MMAU DAR4 was tolerated at doses up to 12 mg/kg in cyno, which represents 2- to 4-fold higher dose than that observed with vedotin ADCs, and had increased terminal half-life and exposure. The optimized trastuzumab-MMAU ADC showed potent antitumor activity and was well tolerated with excellent pharmacokinetics in non-human primates, leading to a superior preclinical therapeutic window. The data supports potential utility of trastuzumab-MMAU for treatment of HER2+ tumors.

Abstract B128: Preclinical characterization of ETx-22, a next-generation antibody drug conjugate (ADC) targeting nectin-4

Abstract Nectin-4 is overexpressed in a variety of cancers including urothelial, breast, cervix, lung and ovarian, with low level of expression in normal tissues. Enfortumab vedotin (EV), an anti-Nectin-4 ADC with a monomethyl auristatin (MMAE) payload, has been a significant advancement in the treatment of urothelial cancer. Yet, it is associated with important skin toxicity owing to the expression of nectin-4 in the skin, and peripheral neuropathy secondary to its toxin. We have developed a next-generation anti-nectin-4 ADC, ETx-22, which consists of a humanized IgG1, Fcg-silent antibody that is bound via maleimide-ß-glucuronide poly-sarcosine linkers to 8 molecules of the topoisomerase-I inhibitor, exatecan. Here, we describe the key pharmacological experiments, in-vivo efficacy and results from the good laboratory practice (GLP) toxicology studies. In-vitro studies have shown that the ETx-22 antibody exhibits a high affinity for tumor-expressed Nectin-4, but 10-fold lower affinity for nectin-4 expressed on skin, with significantly higher internalization in tumor cells. In-vivo efficacy was evaluated in various patient-derived xenograft models. ETx-22 was shown to be consistently superior to EV as well as chemotherapy, in a broad array of tumors, including urothelial, ovarian, cervical and triple negative breast cancer irrespective of the degree of nectin-4 expression. Antitumor activity was also observed in MMAE-resistant models. GLP studies evaluated repeated doses of ETx-22 in cynomolgus monkeys at two doses, 10 and 20 mg/kg. The treatment was administered as 30-minute intravenous infusions once every 2 weeks for a total of three cycles. The ETx-22 antibody alone was also evaluated at 20 mg/kg. Both ETx-22 doses were well tolerated and all animals survived up to scheduled euthanasia. The common observation at both doses was mild multifocal skin hyperpigmentation, which was reversible. No skin rash or pruritus was reported. Mild transient decrease in red blood cells was observed in between cycles. No gastrointestinal toxicities were observed. No signs of interstitial lung disease or eye toxicity were detected. Based on these findings, the highly non- severely toxic dose (HNSTD) is at least 20 mg/kg. The half-life of ETx-22 ranged from 3.36 – 3.6 days. In conclusion, preclinical and pharmacological shows that ETx-22 has better tolerance, longer half-life, and higher efficacy to EV. No skin rash was reported in toxicology studies. A first in human study (EXCEED) is planned to start in 2023. Citation Format: Hatem Azim, Florence Lhospice, Xavier Preville, Joanna Fares, Marc Lopez, Emerence Crompot, Emmanuelle Josselin, Remy Castellano, Maria Wehbeh, Yves Collette, Jack Elands, Daniel Olive. Preclinical characterization of ETx-22, a next-generation antibody drug conjugate (ADC) targeting nectin-4 [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B128.

Quantitation of total antibody (tAb) from antibody drug conjugate (ADC) PYX-201 in rat and monkey plasma using an enzyme-linked immunosorbent assay (ELISA) and its application in preclinical studies

PYX-201 is an investigative ADC oncology drug composed of a monoclonal human immunoglobulin G (IgG) antibody targeting the extra domain B splice variant of fibronectin (EDB + FN) conjugated to an auristatin payload through a cleavable linker. Effective measurement of PYX-201 tAb is the key to ADC drug PYX-201 preclinical pharmacokinetics (PK) assessment. PYX-201 monoclonal antibody (mAb) was used as the reference standard, goat anti-human IgG polyclonal antibody (pAb) or rabbit anti-human Kappa light chain mAb was employed as the capture antibody, and mouse mAb or goat pAb anti-human IgG the crystallizable fragment (Fc) (horseradish peroxidase (HRP)) was utilized as the detection antibody in this ELISA. This assay was validated with a dynamic range 250 – 10,000 ng/mL and 250 – 6000 ng/mL in rat and monkey K2EDTA plasma, respectively. PYX-201 tAb bioanalytical ELISA assay was reported for the first time in any biological matrix. This is the first time for a bioanalytical method to be validated for a tAb from an ADC drug targeting EDB + FN in any biological matrix.

Abstract 2633: Zanidatamab zovodotin (ZW49) induces hallmarks of immunogenic cell death and is active in patient-derived xenograft models of gastric cancer

Abstract Zanidatamab zovodotin (ZW49) is an antibody drug conjugate (ADC) comprised of a bispecific anti-HER2 IgG1 antibody (ZW25, zanidatamab) conjugated to a microtubule inhibitor auristatin payload (ZD02044) via a protease cleavable linker. Clinical studies of ZW49 in HER2-expressing advanced solid tumors are currently underway (NCT03821233). We have previously presented data illustrating the rapid internalization of ZW49 and the anti-tumor activity of ZW49 in multiple patient-derived xenograft models originating from breast carcinomas. Here we present ZW49’s ability to induce in vitro hallmarks of immunogenic cell death (ICD) including increased calreticulin exposure, increased high mobility group box-1 (HMGB1) exposure, and increased extracellular ATP secretion in a HER2 dependent manner. Additionally, we present the results from a study evaluating the anti-tumor activity of ZW49 in a panel of patient-derived xenografts of gastric cancer. HER2-positive SK-BR-3 and HER2-negative MDA-MB-468 tumor cell lines were treated with ZW49 and cell surface calreticulin was assessed by flow cytometry. ZW49 treatment of SKBR-3 cells induced a significantly higher percentage of cells staining positive for cell surface calreticulin compared to untreated cells. In contrast, ZW49 treatment of MDA-MB-468 cells resulted in no significant difference in the percentage of cells staining positive for calreticulin compared to untreated cells. ZW49 treated HER2-positive cancer cell lines SK-BR-3 and SK-OV-3 induced higher levels of extracellular ATP compared to untreated cells. In the HER2-negative cell line MDA-MB-468, treatment with ZW49 did not alter levels of extracellular ATP compared to untreated cells. ZW49 induced higher levels of HMGB1 in the HER2-positive cancer cell lines SKBR3 and N87 compared to untreated cells, whereas in the HER2-negative cell line MDA-MB-468, ZW49 induced similar levels of HMGB1 to untreated cells. Anti-tumor activity was observed in 5/7 (71%) PDX models of gastric cancer after a single i.v. dose of 6 mg/kg, including in models with moderate and weak HER2 expression. The strong anti-tumor activity of ZW-49 in vivo, together with its ability to induce ICD and potential adaptive immune responses, support ZW49 as a promising ADC for the treatment of HER2-expressing cancers warranting further investigation, including potential combination with checkpoint inhibitors. Citation Format: Stuart D. Barnscher, Andrea Hernández Rojas, Kevin J. Hamblett, Nichole Escalante. Zanidatamab zovodotin (ZW49) induces hallmarks of immunogenic cell death and is active in patient-derived xenograft models of gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2633.

Initial Phase I Safety Study of Gedatolisib plus Cofetuzumab Pelidotin for Patients with Metastatic Triple-Negative Breast Cancer.

The PI3K pathway is dysregulated in the majority of triple-negative breast cancers (TNBC), yet single-agent inhibition of PI3K has been ineffective in TNBC. PI3K inhibition leads to an immediate compensatory upregulation of the Wnt pathway. Dual targeting of both pathways is highly synergistic against TNBC models in vitro and in vivo. We initiated a phase I clinical trial combining gedatolisib, a pan-class I isoform PI3K/mTOR inhibitor, and cofetuzumab pelidotin, an antibody-drug conjugate against the cell-surface PTK7 protein (Wnt pathway coreceptor) with an auristatin payload. Participants (pt) had metastatic TNBC or estrogen receptor (ER) low (ER and PgR < 5%, HER2-negative) breast cancer, and had received at least one prior chemotherapy for advanced disease. The primary objective was safety. Secondary endpoints included overall response rate (ORR), clinical benefit at 18 weeks (CB18), progression-free survival (PFS), and correlative analyses. A total of 18 pts were enrolled in three dose cohorts: gedatolisib 110 mg weekly + cofetuzumab pelidotin 1.4 mg/kg every 3 weeks (n = 4), 180 mg + 1.4 mg/kg (n = 3), and 180 mg + 2.8 mg/kg (n = 11). Nausea, anorexia, fatigue, and mucositis were common but rarely reached ≥grade 3 severity. Myelosuppression was uncommon. ORR was 16.7% (3/18). An additional 3 pts had stable disease (of these 2 had stable disease for >18 weeks); CB18 was 27.8%. Median PFS was 2.0 months (95% confidence interval for PFS: 1.2-6.2). Pts with clinical benefit were enriched with genomic alterations in the PI3K and PTK7 pathways. The combination of gedatolisib + cofetuzumab pelidotin was well tolerated and demonstrated promising clinical activity. Further investigation of this drug combination in metastatic TNBC is warranted.

Abstract P245: Synergistic effect of combination of pemigatinib with enfortumab vedotin (EV) in human bladder cancer models

Abstract Bladder cancer is among the most prevalent cancers worldwide, with urothelial carcinoma accounting for approximately 90% of cases. Until recently, poor overall survival after chemotherapy combined with immune checkpoint blockade therapies for patients with advanced urothelial carcinoma highlighted the need for new therapies. Although data supports the use of targeted therapies, such as FGFR inhibitors, recent advances in the treatment of bladder cancer are changing the landscape. Enfortumab vedotin (EV) is an antibody drug conjugate (ADC) consisting of an anti-Nectin-4 antibody conjugated to monomethyl auristatin E, which specifically delivers the auristatin payload to Nectin-4 high-expressing urothelial carcinomas. While activity and durability of response from both monotherapy trials as well as combination with pembrolizumab are favorable, additional treatment options might be required for either patients not responding to treatment or those who might develop resistance. Importantly, FGFR activation through rearrangement or mutation is frequently found in the luminal papillary subtype which also expresses high levels of Nectin-4. Thus, we sought to test the potential of combining pemigatinib and EV in models of human bladder cancer which express both activated FGFR3 and Nectin-4. Our initial analysis show that FGFR3 mutant tumors express high levels of Nectin-4. We further demonstrated that two bladder cancer cell lines RT112/84 (FGFR3-TACC3 fusion) and UM-UC-14 (FGFR3S249C) are sensitive to both pemigatinib and EV in vitro and in vivo. Notably, synergistic anti-tumor effects were observed when pemigatinib was combined with EV in vivo. In addition, combination of pemigatinib with EV significantly improved overall survival when compared to single treatments in these models. Altogether, our data strongly suggest a potential for combination of these therapies in the clinic. Citation Format: Rodrigo A. Hess, Lisa Truong, Antony Chadderton, Michelle Frascella, Leslie Hall, Holly Koblish. Synergistic effect of combination of pemigatinib with enfortumab vedotin (EV) in human bladder cancer models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P245.

Uplift (ENGOT-ov67): A pivotal cohort to evaluate XMT-1536 (upifitamab rilsodotin), a NaPi2b-directed antibody drug conjugate for platinum-resistant ovarian cancer.

TPS5607 Background: XMT-1536 (upifitamab rilsodotin), is a first-in-class Dolaflexin ADC targeting NaPi2b, a sodium-dependent phosphate transport protein, broadly expressed in solid tumors such as serous epithelial ovarian cancer (OC) and non-small cell lung adenocarcinoma. XMT-1536 uses the Dolaflexin platform to deliver approximately 10 DolaLock auristatin payload molecules per antibody and is being evaluated in a Phase I study (NCT03319628). Observation of preliminary antitumor activity was reported in the ovarian cancer expansion cohort, including in patients previously treated with bevacizumab and PARPi (Tolcher et al, ASCO 2019; Richardson et al, ASCO 2019; Hamilton et al, ESMO 2020). Updated data on the OC cohort included 31 patients with higher NaPi2b expression as of December 2020 (Mersana Therapeutics, 2021). In these patients, the ORR was 32% and the DCR was 74%. Complete responses were observed in 2 patients with platinum-resistant ovarian cancer, both of whom had received prior treatment with bevacizumab and PARP inhibitors. Platinum resistant ovarian cancer remains a serious unmet medical need as treatment options are limited and response rates to these treatments are low. Based on the favorable safety and efficacy profile of XMT-1536, UPLIFT was designed as a Phase 2 single-arm registrational cohort of patients with platinum resistant ovarian cancer as part of the ongoing Phase I FIH dose escalation and expansion study to accelerate development and provide a streamlined pathway to regulatory review. Methods: The UPLIFT cohort is enrolling patients with platinum resistant high grade serous ovarian, fallopian tube and primary peritoneal cancer with up to 4 prior lines of therapy. The RP2D of XMT-1536 was determined to be 43 mg/m2 administered intravenously every 4 weeks (q4w) and will be the dose evaluated in the UPLIFT cohort. UPLIFT will enroll approximately 180 patients with platinum-resistant advanced ovarian cancer to obtain approximately 100 patients with higher NaPi2b expression. Prior bevacizumab is required for those patients with 1 or 2 prior lines of therapy. Tumor samples (fresh or archived) will be collected prior to enrollment for retrospective tumor tissue evaluation of NaPi2b expression. The primary objective is assessment of confirmed objective response rate to XMT-1536 as assessed by Investigator in patients with higher NaPi2b expression. Secondary endpoints include confirmed objective response rate regardless of NaPi2b expression, duration of response, and adverse events. Correlative aims include assessing blood and tissue biomarkers for association with clinical benefit. This study is being conducted in collaboration with ENGOT and GOG. Patients will be enrolled globally. Clinical trial information: NCT03319628.

Abstract PS10-26: An initial safety study of gedatolisib plus cofetuzumab pelidotin for metastatic triple-negative breast cancer

Abstract Background: The PI3K pathway is dysregulated in the majority of triple-negative breast cancer (TNBCs), yet single agent inhibition of PI3K in TNBC has minimal clinical activity. We previously reported that PI3K inhibition leads to an immediate compensatory up-regulation of the Wnt pathway. Dual targeting of both pathways is highly synergistic against TNBC models in vitro and in vivo. We initiated a Phase I clinical trial of gedatolisib, a pan-class I isoform PI3K/mTOR inhibitor, and cofetuzumab pelidotin, an antibody-drug conjugate against the cell-surface PTK7 protein (Wnt pathway co-receptor) with an auristatin payload. PTK7 is up-regulated after PI3K inhibition and auristatin is in itself synergistic with gedatolisib providing the potential for a dual mechanism of synergy. Methods: Dose escalation proceeded using a traditional 3+3 schema with a small expansion cohort at the final dose level to better characterize safety. Participants had metastatic TNBC or ER low (ER and PgR &amp;lt;5%, HER2 negative) breast cancer, and had received at least one prior line of chemotherapy. The primary objective was safety. Secondary endpoints included objective response (ORR), clinical benefit at 18 weeks (CB18), and progression-free survival (PFS). Exploratory analyses probed the association of tumor DNA, RNA, and IHC with clinical efficacy to identify putative biomarkers. Results: Between January 2018 and January 2020, 18 patients were enrolled in 3 dose cohorts: gedatolisib (qw) &amp; cofetuzumab pelidotin (q3w) 110mg+1.4mg/kg (n=4), 180mg+1.4mg/kg (n=3), and 180mg+2.8mg/kg dose levels (n=11). The median age was 53 years (32-77). Nausea (n=16), anorexia (n=13), fatigue (n=12), and mucositis (n=12) were common but rarely reached &amp;gt;Grade 3 severity (nausea, n=1; fatigue, n=2). Myelosuppression was uncommon (Grade &amp;gt;3 neutropenia, n=2). 16 participants were evaluable for response. 3 achieved a confirmed partial response (PR), and 3 had stable disease (SD). ORR=18.8%. All 3 PRs lasted &amp;gt; 6 months. CB18=31.3%. Median PFS was 2.0 months (95% CI for PFS:1.2-6.2); median OS was 9.5 months (95% CI for OS:4.3-not reached). Correlative analyses of genomic, transcriptomic, and protein biomarkers with response are currently ongoing. Conclusions: The combination of gedatolisib + cofetuzumab pelidotin for the treatment of metastatic TNBC was found to be well tolerated and demonstrated promising clinical activity. Further investigation of this drug combination in metastatic TNBC is warranted. Trial Registration: NCT03243331 Citation Format: Milan Radovich, Jeffrey P. Solzak, Bradley A. Hancock, Sunil Badve, Anna Maria V. Storniolo, Tarah J. Ballinger, Bryan P. Schneider, Kathy D. Miller. An initial safety study of gedatolisib plus cofetuzumab pelidotin for metastatic triple-negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS10-26.

PF-06804103, A Site-specific Anti-HER2 Antibody-Drug Conjugate for the Treatment of HER2-expressing Breast, Gastric, and Lung Cancers.

The approval of ado-trastuzumab emtansine (T-DM1) in HER2+ metastatic breast cancer validated HER2 as a target for HER2-specific antibody-drug conjugates (ADC). Despite its demonstrated clinical efficacy, certain inherent properties within T-DM1 hamper this compound from achieving the full potential of targeting HER2-expressing solid tumors with ADCs. Here, we detail the discovery of PF-06804103, an anti-HER2 ADC designed to have a widened therapeutic window compared with T-DM1. We utilized an empirical conjugation site screening campaign to identify the engineered ĸkK183C and K290C residues as those that maximized in vivo ADC stability, efficacy, and safety for a four drug-antibody ratio (DAR) ADC with this linker-payload combination. PF-06804103 incorporates the following novel design elements: (i) a new auristatin payload with optimized pharmacodynamic properties, (ii) a cleavable linker for optimized payload release and enhanced antitumor efficacy, and (iii) an engineered cysteine site-specific conjugation approach that overcomes the traditional safety liabilities of conventional conjugates and generates a homogenous drug product with a DAR of 4. PF-06804103 shows (i) an enhanced efficacy against low HER2-expressing breast, gastric, and lung tumor models, (ii) overcomes in vitro- and in vivo-acquired T-DM1 resistance, and (iii) an improved safety profile by enhancing ADC stability, pharmacokinetic parameters, and reducing off-target toxicities. Herein, we showcase our platform approach in optimizing ADC design, resulting in the generation of the anti-HER2 ADC, PF-06804103. The design elements of identifying novel sites of conjugation employed in this study serve as a platform for developing optimized ADCs against other tumor-specific targets.

Phase I expansion study of XMT-1536, a novel NaPi2b-targeting antibody-drug conjugate (ADC): Preliminary efficacy, safety, and biomarker results in patients with previously treated metastatic ovarian cancer (OC) or non-small cell lung cancer (NSCLC).

3549 Background: XMT-1536 is a first-in-class ADC targeting the sodium-dependent phosphate transport protein NaPi2b, broadly expressed in NSCLC and ovarian cancer. XMT-1536 utilizes the Dolaflexin platform to deliver 10-12 DolaLock auristatin payload molecules per antibody. In the dose-escalation portion of the Phase I study (NCT03319628), XMT-1536 showed clinical activity at doses &gt;20mg/m2 with confirmed responses and prolonged stable disease in heavily pretreated OC and NSCLC patients, without preselection for NaPi2b expression. XMT-1536 was generally well-tolerated without the severe toxicities observed with other ADC platforms such as neutropenia, peripheral neuropathy, or ocular toxicity (Tolcher et al., ASCO 2019; Richardson et al., SGO 2020). Here, we report on the expansion (EXP) cohort, which included patients with fewer prior lines of therapy, in the ongoing Phase I study. Methods: Doses administered intravenously every 4 weeks (q4w) of 36 and 43 mg/m2 were evaluated in two cohorts (1) high grade serous ovarian, fallopian tube, or primary peritoneal cancer (OC) with up to 4 prior lines of therapy and (2) NSCLC adenocarcinoma; prior treatment with a platinum-based therapy, immune checkpoint inhibitor, and TKI, if indicated. Archival tumor tissue and tissue from a new tumor biopsy were required for retrospective evaluation of NaPi2b expression. Results: As of 10 February 2020, 23 patients (19 OC and 4 NSCLC) were enrolled in the EXP cohort: 16 dosed at 36 mg/m2 and 7 dosed at 43 mg/m2. Adverse events were generally similar to those previously reported, including transient AST elevation, fatigue, nausea, and pyrexia. Clinical responses and stable diseases have been observed. Efficacy data (objective response rate) and initial correlation of NaPi2b score with clinical response will be reported. Available data from all patients with data cutoff in May 2020 will be included. Conclusions: Overall, XMT-1536 treatment demonstrated clinical activity in high grade serous ovarian cancer and NSCLC adenocarcinoma and was generally well-tolerated with no new safety signal trends identified in the EXP. Clinical efficacy and the relevance of NaPi2b expression for treatment with XMT-1536 will be presented. Clinical trial information: NCT03319628 .

Abstract 4035: Protein tyrosine kinase 7 (PTK7) biomarker analysis in patients (pts) treated with PF-06647020, a PTK7 antibody-drug conjugate (ADC), in a phase I dose expansion study

Abstract Background: PF-06647020, an ADC comprising a humanized monoclonal antibody against PTK7, a cleavable valine-citrulline linker, and an auristatin payload, is being investigated in an ongoing Phase I clinical trial in pts with advanced solid tumors. We hypothesized that response to a PTK7-directed ADC would correlate with PTK7 expression in the pts tumor. We report preliminary baseline PTK7 expression in pts tumors and clinical response using a novel CLIA validated laboratory developed test (LDT). Methods: We developed and validated an LDT comprising an anti-PTK7 immunohistochemistry assay with digital tissue analysis using Flagship Biosciences Inc’s cTA® platform to detect membrane-associated PTK7 in formalin-fixed paraffin embedded (FFPE) epithelial malignancies of ovary, breast and lung. Tumor samples were immunolabeled with the LDT and image analysis was applied to derive a digital H-score. Specifically the digital image annotation included desired regions of analysis (ROA) and excluded regions not of interest. The cTA® platform identified each cell in a given ROA and quantified an overall H-score for malignant epithelial cell membranes immunolabeled for PTK7. Non-small cell lung cancer (NSCLC) pts with H-scores ≥ 56.8 were eligible for enrollment whereas ovarian cancer (OVCA) pts were enrolled unselected. Triple negative breast cancer (TNBC) pts were initially enrolled unselected, but after review a requirement for H-scores ≥ 92.8 was implemented for enrollment. Results: PTK7 IHC was performed on baseline FFPE samples from 69 pts and digital H-scores were generated (Table 1). An additional 7 pt samples were not amenable to analysis with the cTA® platform. Conclusions: A novel CLIA validated LDT was developed and implemented to assess PTK7 baseline levels in FFPE tumors from pts treated with PF-06647020. Clinical responses tended to correlate with H-scores that were higher than the mean for each tumor type. Table 1.PTK7 digital H-scores in Phase I dose expansionNumber of SamplesMinimum H-scoreMaximum H-scoreMean H-scoreMean H-score of Non-respondersMean H-score of RespondersOVCA31120089.280.2111.2NSCLC1993287167.8159.2200.3TNBC1953258159.2147.0224.3 Citation Format: Amy Jackson-Fisher, Navi Mehra, Roberto Gianani, Pamela Whalen, Pamela Vizcarra, Shibing Deng, Stephen McComish, Xiaohua Xin, Eric L. Powell. Protein tyrosine kinase 7 (PTK7) biomarker analysis in patients (pts) treated with PF-06647020, a PTK7 antibody-drug conjugate (ADC), in a phase I dose expansion study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4035.

Abstract OT3-06-02: An initial safety study of gedatolisib plus PTK7-ADC for metastatic triple-negative breast cancer

Abstract Background: The PI3K pathway is dysregulated in the majority of triple-negative breast cancer (TNBCs). Contrary to the theory of oncogene addiction, single agent inhibition of the PI3K pathway in TNBC has had only modest activity. Our group has demonstrated preclinically that when PI3K is inhibited, an immediate compensatory up-regulation of the Wnt pathway occurs. The Wnt pathway is known known for its role in cancer metastases and can confer resistance to initial PI3K inhibition. Simultaneous dual targeting of both pathways is highly synergistic against TNBC models in vitro and in vivo. We have initiated a Phase I clinical trial using Gedatolisib (PI3K/mTOR inhibitor) and PTK7-ADC (Wnt pathway) for patients with metastatic TNBC (NCT03243331). Gedatolisib is a pan-class I isoform PI3K/mTOR inhibitor, and PTK7-ADC is an antibody-drug conjugate against the cell-surface PTK7 protein (Wnt pathway co-receptor) with an Auristatin payload. PTK7 is an attractive second target due to its up-regulation after PI3K inhibition and its known overexpression in TNBC. Further data has shown that the PTK7-payload, Auristatin, is in itself synergistic with Gedatolisib. The combination of using both of these drugs suggests a unique concept of “double synergy”. Where Gedatolisib increases the expression of the target of PTK7-ADC leading to one mechanism of synergy, and the Auristatin payload on PTK7-ADC is synergistic with Gedatolisib providing a second mechanism. Study Design: This is an open-label, Phase I, dose-escalation study with a 3 + 3 cohort design. The trial will enroll 12-18 patients. 3 cohorts of at least 3 patients will receive Gedatolisib (weekly) &amp; PTK-ADC (q3w) at 110mg+1.4mg/kg, 180mg+1.4mg/kg, and 180mg+2.8mg/kg dose levels. Eligibility Criteria: This trial enrolls patients with metastatic triple negative (ER-, PgR-, HER2-) or low estrogen expressing (ER and PgR &amp;lt;5%, HER2-) breast cancer. Patients must have received at least one prior chemotherapy for advanced disease and have adequate hematologic, renal, and hepatic function. Patients with previously treated CNS involvement are eligible. Patients with uncontrolled diabetes are excluded, given the potential for hyperglycemia with Gedatolisib. Patients must have disease amenable and consent to biopsy for correlative endpoints. Objectives: The primary objective is to evaluate the safety of Gedatolisib plus PTK7-ADC. The secondary objective is to evaluate efficacy as determined by objective response rate, clinical benefit at 18 weeks, and progression free survival (PFS). Exploratory objectives will evaluate efficacy in patients with genomic aberrations in the PI3K pathway; and association of tumor DNA, RNA, plasma and circulating tumor cell sequencing with clinical efficacy to identify putative biomarkers. Correlative Sciences: We are collecting matched pre-/post-treatment tumor biopsies and serial blood samples to determine biomarkers of clinical response to inform subsequent trials. We plan to evaluate: 1) PI3K activity; 2) genomic aberrations in the PI3K pathway; 3) baseline PTK7 expression; 4) PTK7 upregulation after Gedatolisib treatment; and 5) mutations in plasma circulating tumor DNA. Supported by the BCRF, 100 Voices of Hope, Catherine Peachey Foundation, and Pfizer. Citation Format: Radovich M, Solzak JP, Hancock BA, Storniolo AMV, Schneider BP, Miller KD. An initial safety study of gedatolisib plus PTK7-ADC for metastatic triple-negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr OT3-06-02.

Abstract P6-17-13: ZW49, a HER2 targeted biparatopic antibody drug conjugate for the treatment of HER2 expressing cancers

Abstract Background: HER2-targeted therapies have transformed the treatment of patients with HER2-expressing breast and gastric cancers. Despite this, there remains a need for new treatments that are well tolerated and effective not only for cancers with high HER2 expression levels but also those with lower levels of expression. ZW49 is an antibody drug conjugate (ADC) that combines a novel auristatin payload (potent anti-cancer agent) with the unique mechanisms of action of the anti-HER2 biparatopic antibody, ZW25, which binds to the same domains as trastuzumab and pertuzumab. In preclinical studies in cancer cell lines with low to high levels of HER2 expression, ZW25 is associated with increased binding and internalization compared to trastuzumab, while the novel N-acyl sulfonamide auristatin payload has demonstrated increased in vivo tolerability compared to other microtubule inhibitors. ZW49 therefore has the potential to address unmet medical need across a range of HER2-expressing cancers. Methods: Multiple in vitro and in vivo experiments were performed to characterize ZW49 as a potential therapeutic candidate. Internalization and cell growth inhibition of ZW49 were evaluated in HER2-expressing cell lines. Anti-tumor activity was assessed in patient-derived xenograft (PDX) tumor models of HER2 low and high expressing breast cancers. Tolerability was assessed in a 6-week repeat-dose non-GLP toxicology study in non-human primates (NHP) with intravenous administration of ZW49 at either 9 mg/kg or 12 mg/kg once every two weeks. Results: In vitro, ZW49 was more rapidly internalized into HER2-expressing cells compared to a monospecific trastuzumab-ADC. ZW49 also displayed potent in vitro cell growth inhibition in several breast cancer cell lines with a range of HER2 expression. This activity was confirmed in multiple in vivo PDX models, including a HER2 IHC 3+ HBCx-13b xenograft where two doses of ZW49 at 3 mg/kg or higher generated tumor regressions, and a HER2 IHC 1+ ST-910 xenograft where a single dose of ZW49 at 6 mg/kg or higher generated regressions. In both models, regressions occurred at exposures that were well tolerated in NHP. In a repeat dose pilot non-GLP study the highest dose tested (12 mg/kg) was considered to be the no observed adverse effect level (NOAEL) and a GLP repeat-dose toxicology study was ongoing at the time of abstract submission. Conclusions: ZW49 is a novel biparatopic HER2-targeted ADC that demonstrated anti-tumor activity in low and high HER2-expressing breast cancer cell lines and PDX models. Notably, tumor regressions were observed at exposure levels that were well tolerated in NHP. These results support the potential of ZW49 as a novel therapeutic agent that may help address unmet medical need in patients with high and low HER2-expressing cancers. Citation Format: Hamblett KJ, Barnscher SD, Davies RH, Hammond PW, Hernandez A, Wickman GR, Fung VK, Ding T, Garnett G, Galey AS, Zwierzchowski P, Clavette BC, Winters GC, Rich JR, Rowse GJ, Babcook JS, Hausman D. ZW49, a HER2 targeted biparatopic antibody drug conjugate for the treatment of HER2 expressing cancers [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-17-13.

Abstract 1978: Antibody-drug conjugates with DNA inhibitor linker-payloads can overcome resistance in cultured tumor cells made resistant to conjugates with cleavable-linked auristatins

Abstract Antibody drug conjugates (ADCs) are targeted biotherapeutics delivering cytotoxic payloads to tumor-expressed antigens. The most common class of ADC payloads are microtubule inhibitors (MTI) and DNA inhibitors, exemplified by clinically approved ado-trastuzumab emtansine (TDM1; MTI DM1), brentuximab vedotin (MTI auristatin), gemtuzumab ozogamicin and inotuzumab ozogamicin (DNA double strand breaker calicheamicin). Auristatin payloads are in active development, primarily employing proteolytically cleavable linker mcValCitPABC. There are many benefits of cleavable-linked permeable payloads, including bystander activity in a heterogeneous tumor environment. However, due to the complexity of human cancer, tumors become refractory to most drug therapies. We hypothesized that cultured tumor cells chronically treated with mcValCitPABC-auristatin ADCs would acquire varied resistance mechanisms. As model ADCs, we used trastuzumab-mcValCitPABC-mono methyl auristatin E (TvcMMAE) and trastuzumab-mcValCitPABC-Aur0101 (Tvc0101). Her2 expressing gastric carcinoma line, N87, was chronically exposed to the respective ADCs at cytotoxic doses in a cyclical fashion (3 days on; ~1 - 3 weeks off). Resistance was acquired very slowly (over ~1 year), and cells were characterized for their resistance profile. N87-TvcMMAE cells had &amp;gt;1000X relative resistance (RR) to T-auristatin and TDM1 ADCs compared with parental N87, and showed moderate to high resistance (~50 – 300X) to unconjugated MTIs including auristatins and DM1. The cells were cross-resistant (&amp;gt;100 - 1000X) to T-ADCs delivering several payload classes, including NAc-calicheamicin and cyclopropylpyrroloindolone (CPI). Her2 and MDR1 levels were unchanged in N87-TvcMMAE cells, however, a slight increase of MRP1 was observed on immunoblots. Cells made resistant to Tvc0101 ADC showed a different profile. N87-Tvc0101 cells had &amp;gt;1000X RR to T-auristatin ADCs and &amp;gt;400X to TDM1, but were sensitive (&amp;lt;4X) to unconjugated auristatins and DM1. Interestingly, these cells retained sensitivity to T-ADCs delivering DNA inhibitor payloads, including NAc-calicheamicin (&amp;lt;5X) and CPI (&amp;lt;14X). These data are striking since N87-Tvc0101 cells had significantly reduced Her2 levels, yet responded to other Her2 ADCs. Immunoblots indicated no observed changes in MDR1 or MRP1 protein. The cross-resistance profile of each cell model is distinct, despite using the same parental cell background and ADCs differing in the auristatin. These data suggest that different resistance mechanisms develop upon treatment with structurally similar mcValCitPABC-auristatin ADCs, and that resistant tumors may respond to ADCs delivering a different linker-payload yet targeting the same antigen, even in cases where antigen levels have decreased. Citation Format: Xingzhi Tan, Matthew Sung, Frank Loganzo. Antibody-drug conjugates with DNA inhibitor linker-payloads can overcome resistance in cultured tumor cells made resistant to conjugates with cleavable-linked auristatins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1978.

Abstract 3912: Towards development of next-generation biparatopic ADCs using a novel linker-toxin with expanded therapeutic window

Abstract Antibody drug conjugate (ADC) therapies such as Kadcyla® and Adcetris® have significantly improved outcomes for patients. Despite these early advances, many ADCs have failed due to tolerability and efficacy concerns; therefore, there is a need to develop ADCs with a greater therapeutic window. We have previously reported the increased tolerability of a novel N-acyl sulfonamide auristatin payload conjugated to trastuzumab via a protease cleavable linker. In non-human primates (NHPs), the HNSTD for this ADC was 18 mg/kg compared to 3 mg/kg for the MMAE conjugate control. Separately, we have also reported that a biparatopic antibody targeting a tumor associated antigen (e.g. anti-HER2 bispecific antibody ZW25) can lead to enhanced receptor clustering and improved internalization, thereby increasing the efficiency of payload delivery. Our aim is to develop a series of novel biparatopic ADCs with expanded therapeutic windows against multiple targets. Here we present the proof-of-concept in vitro and in vivo characterization of benchmark ADCs against 3 different targets with improved tolerability and equivalent efficacy. Benchmark antibodies against 3 known clinical targets were conjugated to our N-acyl sulfonamide auristatin (mAb-ADCs) or to MMAE or DM4 controls (mAb-control ADCs) via cleavable linkers and were assessed for in vitro binding affinity and cytotoxicity. The therapeutic windows of mAb-ADCs and mAb-control ADCs were compared by assessing efficacy in mouse xenograft models and tolerability and pharmacokinetics in NHPs. mAb-ADCs had similar binding affinities to recombinant targets and/or to cancer cells expressing low to high levels of target antigen compared to the mAb-control ADCs. mAb-ADCs demonstrated similar in vitro cytotoxicity compared to mAb-control ADCs and this was recapitulated in vivo with similar tumor growth inhibition in mouse xenograft models. In a NHP tolerability/PK study, mAb-ADCs for all 3 targets were tolerated at doses up to 18 mg/kg (single dose IV infusion) compared to the mAb-control ADCs that showed severe to life-threatening neutropenia at lower doses. The increase in maximum tolerated dose for the mAb-ADCs over the mAb-control ADCs, together with comparable efficacy across 3 different targets, demonstrates the broad applicability of the novel N-acyl sulfonamide auristatin payload to expand the therapeutic window. This strategy, together with ongoing efforts to identify synergistic antibody paratopes that more efficiently deliver payload, could lead to next-generation biparatopic ADCs with improved activity. Citation Format: Rupert H. Davies, Stuart D. Barnscher, Peter W. Chan, Laurence Madera, Jamie R. Rich, Marylou Vallejo, Grant R. Wickman, Kevin Yin, Vincent Fung, Kevin J. Hamblett, Patrick G. Kaminker, John S. Babcook. Towards development of next-generation biparatopic ADCs using a novel linker-toxin with expanded therapeutic window [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3912.

Abstract 3914: ZW49, a HER2-targeted biparatopic antibody-drug conjugate for the treatment of HER2-expressing cancers

Abstract Therapies targeting HER2 have transformed the treatment of patients with HER2-expressing breast and gastric cancers. Unfortunately, many patients recur following HER2-targeted treatments and new therapies are needed. Multiple antibody-drug conjugate (ADC) technologies are being explored in this setting, some of which utilize the anti-HER2 antibody trastuzumab. Here we present the preclinical characterization of a new anti-HER2 biparatopic ADC, ZW49, which is generated from the conjugation of a novel N-acyl sulfonamide auristatin payload to the inter-chain disulfide bond cysteines of the bispecific anti-HER2 IgG1 antibody ZW25, via a protease cleavable linker. A series of in vitro and in vivo experiments were performed to characterize ZW49 as a potential therapeutic candidate. In cellular binding assays, it was confirmed that the payload conjugation to ZW25 did not affect the antibody's binding to HER2-expressing cells. ZW49 displayed potent in vitro cytotoxicity in multiple cancer cell lines expressing HER2 and was efficacious in multiple patient-derived xenograft (PDX) models. In mice bearing the HBCx-13b HER2 3+ PDX, two doses of ZW49 administered two weeks apart generated tumor regressions. Furthermore, preliminary results from PDX models with lower levels of HER2 expression treated with ZW49 also generated regressions. In nonhuman primates ZW49 administered intravenously every two weeks for three doses was well tolerated. Based on these findings, we are proceeding with further development of ZW49 as a therapeutic candidate in HER2-expressing cancers. Citation Format: Kevin J. Hamblett, Phil W. Hammond, Stuart D. Barnscher, Vincent K. Fung, Rupert H. Davies, Grant R. Wickman, Andrea Hernandez, Tong Ding, Adam S. Galey, Geoffrey C. Winters, Jamie R. Rich, John S. Babcook. ZW49, a HER2-targeted biparatopic antibody-drug conjugate for the treatment of HER2-expressing cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3914.

Clinical pharmacology assessment of PF-06647020 (PF-7020), an antibody-drug conjugate (ADC) targeting protein tyrosine kinase 7 (PTK7), in adult patients (pts) with advanced solid tumors.

2574Background: PF-7020, an ADC composed of humanized monoclonal antibody (Ab) against PTK7, auristatin payload, and valine-citrulline linker, is being investigated in the ongoing first in human Ph...